A Novel Prognostication System for Spinal Metastasis Patients Based on Network Science and Correlation Analysis.

AIMS: During the progress of oncological diseases, there is an increased probability that spinal metastases may develop, requiring personalised treatment options. Risk calculator systems aim to provide assistance in the therapeutic decision-making process by estimating survival chances. The predictive ability of such calculators can be improved, thereby optimising the choice of personalised therapy. The aim of this research was to create a new risk assessment system and show a method with which other centres can develop their own local score. MATERIALS AND METHODS: We created a database by retrospectively processing 454 patients. The prognostic factors were selected via a network science-based correlation analysis that maximises Uno's C-index, keeping only a small number of predictors. To validate the new system, we calculated the D-statistic, the Integrated Discrimination Index, made a five-fold cross-validation and also calculated the integrated time-dependent Brier score. RESULTS: As a result of multivariate Cox analysis, we found five independent prognostic factors suitable for the design of the risk calculator. This new system has a better predictive ability compared with six other well-known systems with an average C-index of 0.706 at 10 years (95% confidence interval 0.679-0.733). CONCLUSIONS: An accurate estimation of the life expectancy of cancer patients is essential for the implementation of personalised medicine. The training performance of our system is encouraging, indicating the benefit of a network science-based visualisation step. We believe that in order to further improve the prediction ability, it is necessary to systematise previously 'unknown' factors (e.g. radiological morphology).

A histological analysis of gingival condition associated with orthodontic treatment.

The aim of this histological study was to analyze the gingival reaction to fixed orthodontic appliances. Gingival specimens were obtained with minimal trauma from 11 patients treated with fixed appliances in different intervals during the orthodontic treatment, including post-treatment periods. Serial sections were stained with Hematoxylin-Eosin. T- and B-cells were identified by specific antibodies, using a double staining technique with Avidin-Biotin method. Histological observations demonstrated and confirmed the presence of gingivitis during orthodontic treatment. According to the usual histological evaluation, the biopsies revealed the presence of hyperplastic chronic inflammatory changes from mild to moderate severity. The lack of rapid increase of CD20+ cells demonstrated that the gingival inflammation did not cause overall tissue destruction.

The characterization of PDGFR-alpha and PDGFR-beta expression in malignant non-Hodgkin lymphoma.

PDGF receptors play an important role in tumor progression as being part of a group of receptors that are expressed along the membrane of tumor cells. The aim of this study was to evaluate the PDGF receptor expression in follicular and diffuse forms of non-Hodgkin malignant lymphoma. We evaluated 38 biopsy fragments from patients diagnosed with malignant non-Hodgkin lymphoma. We noticed the distribution of PDGFR-alpha and -beta in tumor cells and the immunoreactivity was quantified as the percentage of positive tumor cells. We noticed the presence of score 3, more than 30% of tumor cells positive, for PDGFR-alpha and PDGFR-beta in 50% and 75% cases of follicular non-Hodgkin lymphoma. For diffuse malignant non-Hodgkin lymphomas, score 3 was noted in 23.52% of cases for PDGFR-alpha and 35.29% of cases for PDGFR-beta. This may represent an important therapeutic target in patients who do not respond to conventional therapy, but further research is needed for a careful evaluation of benefits and side effects of PDGFR inhibitors.

The influence of diabetes mellitus on periodontal tissues: a histological study.

OBJECTIVE: The aim of this study was to investigate histological changes that occur in the periodontium of subjects with type 2 diabetes mellitus without signs of periodontal disease and to establish the influence of this systemic condition upon periodontal structures. MATERIALS AND METHODS: Gingival tissue samples were obtained from 12 adult patients with type 2 diabetes mellitus and 10 healthy adults, as control group. The specimens were examined using standard dyes as Hematoxylin and Eosin and PAS-Alcian stain, by a microscope with different magnifications. RESULTS: Our results showed that periodontal disease in patients with type 2 diabetes mellitus is characterized by significant inflammation, affecting both epithelial and connective tissues, with degeneration of dermal papilla, increase in number of inflammatory cells, destruction of reticular fibers and accumulation of dense collagen fibers (fibrosis). CONCLUSIONS: Within the limits of this study, diabetic subjects presented distortion in periodontal attachment, with changes in both epithelial and connective tissues, when compared to the healthy controls, suggesting that diabetes mellitus has an independent effect on periodontal tissue. This effect is observed in both groups, so that we considered it to be independent of the periodontal condition.

Retinal and renal vascular permeability changes caused by stem cell stimulation in alloxan-induced diabetic rats, measured by extravasation of fluorescein.

AIM: To determine whether treatment with the stem cell stimulator Olimpiq® Stem×Cell prevents increase of retinal and renal vascular permeability in alloxan-induced diabetic rats. MATERIALS AND METHODS: Two groups of Wistar rats were made diabetic by single intraperitoneal injection of Alloxan. The third, the control group, received vehicle alone. One diabetic group received Olimpiq® Stem×Cell treatment for 4 weeks. The permeability of the blood-retinal barrier (BRB) and renal vessels were measured by the extravasation of fluorescein-labeled bovine serum albumin. RESULTS: Six weeks subsequently to Alloxan injection, significantly elevated the tissue fluorescence, the renal vascular leakage and BRB breakdown was demonstrated in the diabetic group, compared to the nondiabetic group. Olimpiq® Stem×Cell treatment significantly reduced the BRB breakdown, tissue fluorescence, and vascular leakage. CONCLUSION: Olimpiq® Stem×Cell would be a useful choice of treatment for complications associated with increased vascular permeability of diabetes, such as retinopathy or nephropathy.

Microvascular density in non-Hodgkin B-cell lymphomas measured using digital morphometry.

INTRODUCTION: The growth of solid tumors requires the development of microvessels, therefore tumor expansion depends on angiogenesis. Microvessels provide nutrients and oxygen and remove catabolytic substances, while endothelial cells produce growth factors for tumor cells in a paracrine fashion. The microvascular component of a tumor also plays a role in the metastatic capacity of the tumor, enabling the tumor cells to spread to distant locations by providing a large endothelial surface. AIM: The purpose of this study was to review the literature about angiogenesis regarding malignant lymphomas and to perform basic measurements by means of digital morphometric methods in large B-cell lymphomas and follicular lymphomas. MATERIALS AND METHODS: After thorough analyzing currently available assessment methods, we performed angiogenesis assessment on 19 randomly selected cases, from paraffin-embedded specimens using digital morphometry. We used immunohistochemistry and the CD34 antigen to mark microvessels. We measured average vascular diameter and a previously successfully applied digital morphometric method to quantify the extent of endothelial area. RESULTS: According to literature data, our knowledge and understanding of angiogenesis grew rapidly from early studies such as Folkman's classic paper. Many studies showed that angiogenesis plays a key role in the biology of tumors and therefore the study of angiogenesis might open new therapeutic possibilities. There have been many studies of angiogenesis in malignant lymphomas, however not as many articles as in other tumor types. Our morphometric studies showed there are statistically significant differences between diffuse large cell lymphoma (DLBCL) and follicular lymphoma (FL) regarding average vascular diameter and that high grade lymphomas tend to have a greater CD34+ endothelial area.

Mast cells contribute to the angiogenesis in non-Hodgkin lymphoma. An immunohistochemical study based on the relationship with microvessel density.

Only few data are available in the literature concerning angiogenesis in hematological malignancies. Non-Hodgkin lymphoma classified on the base of molecular profile is frequently characterized by unpredictable behavior that seems to be related to tumor cells and also to the tumor microenvironment. The tumor microenvironment contains blood vessels and a large variety of cells that can play an important role to the progression of angiogenesis and tumor growth. From these, mast cells have been shown to be a source of angiogenic factors. The aim of this work was to investigated the relationships between mast cells and blood vessels in non-Hodgkin lymphoma and reactive lymphoid tissue from three different anatomical sites. Using double immunostaining method CD34/mast cell tryptase we noticed that mast cell density was significantly lower in the follicular lymphoma than in diffuse type lymphoma. The morphology of vessels, the presence of pillars and splitting suggested that intussusceptions is the main mechanism of angiogenesis. In the cases with primary lymphoma of the spleen, we found few mast cells and a high number of blood vessels. Our data suggest that lymphoma-associated angiogenesis is driven in part by the tumor microenvironment, and particularly, by mast cells. On the other hand, our results support the organ-specific tumor-associated angiogenesis in malignant non-Hodgkin lymphoma.

Immunohistochemical comparative study of fibrosis and biliary ductular reaction in alcoholic and viral chronic hepatitis.

Our study includes 102 cases of liver biopsy previously diagnosed with chronic alcoholic hepatitis and also B and C viral hepatitis. In these cases, we analyzed the extension of fibrosis with two different methods. First, we evaluated fibrosis with the subjective Knodell score; secondly, we used digital image analysis to achieve this. We also used immunohistochemical methods to mark those cells positive at Smooth Muscle Actin (SMA) and Glial Fibrillary Acidic Protein (GFAP). We have observed that the extension of fibrosis was most predominant in cases with B viral chronic hepatitis, while the number of cells responsible of fibrosis (stellate cells, myofibroblasts) was highest in C viral chronic hepatitis. These differences help clinician to divide patients into those who may be treated with interferon and those treatable with antiviral therapy. We observed ductular reaction (as shown by cytokeratin 7 immunostaining) within the lobular structure more frequently in alcohol related chronic hepatitis, whilst in C viral chronic hepatitis this reaction was more readily seen in portal spaces. We have concluded that patients with C viral hepatitis can benefit most from a correctly indicated hepatic biopsy since in these cases the lesions might be observed in an early and potentially curable phase.

Diagnostic and differential diagnostic criteria of lymphoid neoplasms in bone marrow trephine biopsies: a study of 87 cases.

The aim of this study is to present the diagnostic and differential diagnostic criteria of the bone marrow specimen involved by lymphomas based on the histomorphological immunophenotype features and clonality of the tumor cells, patterns of lymphoproliferation and diagnostic pitfalls. BMB material obtained from the right posterior iliac crest was represented from 87 untreated and treated patients with BM involving malignant lymphoma, stained with Hematoxylin-Eosin, Giemsa, Periodic Acid Schiff and Gömöri's Silver. In order to perform immunohistochemistry examination we used a large antibody panel. B-cell clonality was determined in six cases. We found eight reactive lymphoproliferative responses and 79 lymphoid neoplasms of which 45 were diagnosed as de novo lymphoma, the rest of 34 samples being examined for staging. The predominant lymphoma was CLL (30 cases), over followed by DLBCL (18 cases). The most frequent patterns of involvement were the interstitial (29%) and mixed (15%) ones. In eight cases, we found reactive lymphoid aggregates. The B-cell clonality test showed four monoclonal, one oligoclonal and one polyclonal diseases form. Diagnosis of lymphoma versus reactive aggregate has been based on the combination of a lot of antibodies and involvement pattern. Although investigation of gene rearrangement was necessary for the establishment of the correct diagnosis in only 6.9% of cases, it should be emphasized that it is of great importance in disease monitoring.

The evaluation of the sentinel lymph nodes status in breast carcinoma using microscopic, immunohistochemical and cytomorphometric methods in order to establish new stadializations and therapeutic schemes.

The state of axillary lymph nodes represents the most important prognostic parameter in patients with breast carcinoma. The biopsy and examination of sentinel lymph nodes, the former one containing metastases originating in mammary carcinoma, allows a better stadialization of the tumor but also the avoiding of the extirpation of the axilla, associated with a series of complications and high costs of hospitalization. In establishing the tumoral prognosis, not only the diameter but also the localization of the metastasis in the lymph nodes is utterly important. The evaluation of the metastases was carried out through the serial examination of the sentinel lymph node correlated to immunohistochemical examinations with AE1/AE3. Of the 570 patients with breast carcinoma evaluated in this research, 250 had macrometastases, 93 micrometastases, only 23 had isolated tumor cells, and in the case of 204 no metastases were found. The technique of computerized cytomorphometry allowed a better evaluation of the diameter and localization of the metastases in the lymph nodes than the examination through optical microscope. The tumoral prognosis in the case of patients with macrometastases is poorer than that of patients with micrometastases. The patients in whom only the presence of isolated tumoral cells was demonstrated have a similar prognosis with those who do not have metastases. As far as the localization of micrometastases in the sentinel lymph nodes is concerned, those with a subcapsular localization are associated with a poorer prognosis than those with an intraparenchymatous localization. As well as this, the subcapsular localization of micrometastases was also associated with the diameter of the primary tumor extending between 2-5 centimeters, a high microscopic grade, the presence of lymph vascular emboli and microscopic type of the primary tumor associated with poor prognosis. On the other hand, the presence of isolated tumoral cells was associated with tumors of a small diameter lacking the presence of lymph vascular emboli and with a low microscopic grade. All these data are essential in establishing the therapeutic management of the patients with breast carcinoma; consequently, we recommend their inclusion in future stadializations of this lesion and the evaluation of tumoral prognosis.

The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

The angiogenesis in colorectal carcinomas with and without lymph node metastases.

UNLABELLED: Many clinical trials revealed that the anti-angiogenic treatment could improve prognosis in patients with metastatic colorectal carcinomas (CRC), when added to standard chemotherapy. In this paper, we tried to find out if the microvascular density (MVD) determined with CD31, CD105 was correlated with lymph node status, and if the intensity of angiogenesis was different in right versus left colon segments. We studied 187 CRC, with and without lymph node metastases, 128 from left and 59 from right colon. RESULTS: In the right colon, the MVD was higher in the cases where the lymph nodes did not present metastases (pN0) but also when four or more lymph nodes were involved (pN2). In the rectum and sigma, the angiogenesis presented the highest intensity in pN0 and pN1 stage (1-3 lymph nodes with metastases), decreasing in pN2 stage. In the descendent colon segment, the MVD did not present differences between the cases with and without lymph node metastases. CONCLUSIONS: Our study reveals that the most indicated cases for antiangiogenic treatment seem to be the pN0 and pN1 cases in the rectum and sigma, respectively pN0 and pN2 cases in the right colon. We tend to believe that the angiogenesis intensity in CRC is higher in early-stages of the tumoral proliferation but it is not an increasing process, having rather an oscillating character. Therefore, the angiogenesis remains an independent prognostic and predictive factor and the antiangiogenic treatment is necessary to be individualized for each patient.

The aspects of angiogenesis in anal canal carcinomas compared with that in colorectal carcinomas.

AIM: To compare the angiogenesis in anal canal carcinomas (ACC) with that in colorectal carcinomas (CRC). METHODS: A number of 507 CRC, surgical specimens, were analyzed, 12 cases (1.97%) being ACC. In 20 cases from left and right colon (CRC) and in the 12 ACC we analyzed the immunohistochemical parameters related to angiogenesis, utilizing the following LabVision antibodies: CD31, CD105 (endoglin) and VEGF1. Morphometrical analysis and positive cell counting were performed in the tumoral and peritumoral tissue. Immunoperoxidase method was used. RESULTS: The average age was 63.17 +/- 10.87 years in CRC, respectively 57.9 +/- 10.05 years in the ACC (p<0.0001). Compared with CRC the ACC occur more frequently at the females (58%). Angiogenesis was expressed in the majority of cases. In CRC, the microvascular density (MVD) was higher than that from ACC. The ratio CD31/CD105 was 1 in ACC and 3 in CRC. VEGF was positive in 25% of ACC and 80% of CRC. In CRC were more mature vessels, marked only with CD31 than immature vessels or endothelial isolated cells marked with both CD31 and CD105. In ACC prevailed the neoformed vessels marked with both CD31 and CD105. CONCLUSIONS: The performed assessments have showed a higher incidence of ACC at females and at younger ages. The angiogenesis in ACC was not so high like in CRC and the immature neoformed vessels was more frequently. In ACC, the antiangiogenic treatment that regards the VEGF inhibition seems to be not as efficient as in CRC. The radiotherapy could stop the angiogenesis and could inhibit the vessels' maturation.

The correlation between the immunostains for p53 and Ki67 with bcl-2 expression and classical prognostic factors in colorectal carcinomas.

UNLABELLED: The prognostic role of p53 and Ki67 in colorectal carcinomas (CRC) is very controversial in the literature. In our study, we tried to find if their immunostains are correlated with bcl-2 expression or other classical prognostic factors (sex, age, localization and size of tumor, the grade and staging of tumor). We studied 507 cases with CRC and chose 38 cases in which we realized these correlations. Fourteen cases were mucinous CRC, the other 24 cases being non-mucinous CRC (six well differentiated, 13 moderate and five poorly differentiated). For statistical analysis, we used the Statistical Program Graph Pad In Stat 3-Trial Version. We considered the significant association when p<0.05, with 95% confidence interval. RESULTS: The median value was 75% for p53 expression, respectively 35% for Ki67 expression. Bcl-2 was positive in 47% of cases but not correlated with p53 or Ki67. We found a significantly statistical decrease p53 immunostain with grade of tumor (70% in well differentiated, respectively 40% in poorly differentiated CRC) and increase of Ki67 median expression (25% in well differentiated, respectively 60% in poorly differentiated CRC). Ki67 was correlated with age of patients, lymph node involvement, being more expressed in N2 (80%) than in N0 (22.5%) and with Dukes MAC staging (25% in B1, 60% in C2). P53 was correlated with age of patients and pT component, after pTNM staging (75% in pT2, 40% in pT4). P53 was not correlated with Ki67. CONCLUSION: The CCR prognostic is not determined only by proliferative capacity of tumoral cells.

Stereochemistry and enantiomeric purity of a novel anxiolytic agent, deramciclane fumarate.

The synthesis, stereostructure, and enantiomeric separation by chromatography of a new, chiral anxiolytic agent, deramciclane fumarate (2, (-)-[1R,2S,4R]-2-(2-dimethylaminoethoxy)-2-phenyl-1,7, 7-trimethylbicyclo[2.2.1]heptane fumarate, EGIS-3886), is described. The optical antipode and the racemate of compound 2 were also prepared. The structure was determined by single crystal X-ray diffraction analysis. The enantiomeric separation was accomplished by HPLC on Chiralcel OD (250 x 4.6 mm; 10 microm) and hexane-ethanol (99.5:0.5) as mobile phase at room temperature. The enantiomeric purity of the synthesized drug substance proved to be very high (>99. 9%). Some statements published earlier on the stereostructure of deramciclane fumarate are critically discussed.

Antiarrhythmic activity of oximethers.

More than sixty 2-substituted-cycloalkanone-oxim-/2-hydroxy-3-dialkylamino/- propylethers were investigated on aconitine induced ventricular arrhythmia model in rats. According to the structure-activity relationships cyclohexanes containing diisopropylamine in the basic group were the most effective derivatives. Based on the highest per os activity and lowest toxicity EGIS-3966 (ED50 values 1.21 mg/kg iv. and 27.3 mg/kg per os) was selected for further development.